There is a liquid chromatograph using a principle of a liquid chromatography in which, to a cylindrical vessel having liquid inlet and outlet in the center of top and bottom disks referred to as a column, a particulate packing having 10 to several hundred microns in particle size referred to as a gel is plugged without allowing for any voids in the space sandwiched between the top and bottom disks intending to achieve a uniform and sufficient contact with the liquids, and then separation and purification of a substance, mainly of a protein, is conducted utilizing the interaction between a solute molecule and the gel upon allowing the liquid to run in a single direction using a pump and the like from any direction from the liquid inlet or outlet on the top or bottom. Currently, a gel for a liquid chromatography having a varying surface structure is marketed. One having an ion exchange group on the surface of a gel has a function as an ion exchange gel, and makes it possible to separate substances by an ion exchange chromatography utilizing an interaction between a solute molecule and an ionic charge.
For example, when using a liquid chromatography, the separation was based on the specificity or the nature of a protein due to a change in the concentration resulting from mixing of two or three solutions. In such a method, a packing is plugged in a column and equilibrated with a buffer with an affinity allowing an intended protein to be adsorbed, and then a sample protein solution dissolved in the same buffer is passed through the column to accomplish the adsorption onto the packing, to which then a buffer solution having a different concentration is applied from the top of the column using a pump and the like, whereby establishing a concentration gradient in the column. At the same time, the concentration gradient in the column is established, and the protein loses an affinity at a concentration corresponding to the affinity of the intended protein, whereby being released from the packing and eluted. Accordingly, when the concentration of the intended protein is unknown, a buffer system allowing for a concentration gradient over a wide range is employed, and after fractionation to a plurality of wells based on the concentration gradient, the absorbance is measured for example by a UV absorption detector to narrow the range, and thereafter a buffer system giving a narrow range of the concentration gradient is employed, whereby extracting the intended protein.
Nevertheless, such a conventional liquid chromatography requires expensive devices and buffer solutions as well as a trial-and-error-based and well-trained operation, and suffers from effects of the concentration and the flow rate of a developing buffer which is not negligible.
Moreover, in such a conventional column, since a liquid is allowed to run in a single direction in a particulate packing plugged in the column, the packing is plugged without allowing for any voids in the column to ensure a high probability of the contact of the packing with a liquid upon passing through it, resulting in the time period of the contact of the liquid with the packing which can be controlled only by the flow rate in the column. However, because of the control of the flow rate using a pump, a transporting medium for moving a liquid containing the intended substance as a processing subject will be required. Thus, a buffer solution will be required in a problematically large quantity which is several times, several ten times, several hundred times as that of the liquid, while the contact with the packing surface will not necessarily high.
In addition, in a conventional column, since a fluid runs only in a single direction and the packing is plugged without any void in a space sandwiched between the disks of the column, a certain fixed route through which the liquid runs in the packing tends to be established. Once such a fixed route is established, a part which can not participate in the reaction may problematically occur.
Also since the mobile phase flow rate or mobile phase concentration has more or less influence, a troublesome sophisticated control is required.    [Patent Document 1] EP No. 198413    [Patent Document 2] International Publication No. 88/09201    [Non-Patent Document 1] Liquid Chromatography Q&A, Itaru Matsushita, GIHODO SHUPPAN Co., Ltd., June, 2000    [Non-Patent Document 2] Practice in Liquid Chromatography, Akira Eto, SANKYO Co., Ltd., 1976